CN115537379B - Combined culture medium for culturing MDCK cells and amplifying viruses and application thereof - Google Patents

Combined culture medium for culturing MDCK cells and amplifying viruses and application thereof Download PDF

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CN115537379B
CN115537379B CN202211523733.4A CN202211523733A CN115537379B CN 115537379 B CN115537379 B CN 115537379B CN 202211523733 A CN202211523733 A CN 202211523733A CN 115537379 B CN115537379 B CN 115537379B
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CN115537379A (en
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陈文庆
赵洪磊
徐舸辰
王杰勇
浦勇
类成霞
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Tianxinhe Suzhou Biotechnology Co ltd
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Abstract

The invention provides a combined culture medium for culturing MDCK cells and amplifying viruses and application thereof, wherein the combined culture medium comprises the following components: culture medium and virus maintenance solution for wall-mounted culture of MDCK cells. The culture medium for the adherent culture of the MDCK cells can quickly finish domestication adaptation of the MDCK cells, supports high-density adherent culture of the MDCK cells in a fixed bed bioreactor, has high cell activity rate and low caking rate, can obtain more excellent performance than serum culture on multiple indexes, improves virus infection capability, and is beneficial to virus infection. The virus after infecting cells can be effectively amplified in the virus maintenance solution, and the virus titer is improved, so that the virus can be better applied to the amplification production of vaccine viruses, and the production efficiency is improved.

Description

Combined culture medium for culturing MDCK cells and amplifying viruses and application thereof
Technical Field
The present invention relates to the field of biotechnology. In particular, the invention relates to a combined culture medium for culturing MDCK cells and amplifying viruses and application thereof.
Background
A virus (Biological virus) is a tiny, structurally simple, non-cellular organism that contains only one nucleic acid (DNA or RNA), must be parasitic in living cells and proliferate in a replicative manner, and is also a common pathogen. Since viruses have no cellular structure, they consist only of the genetic material nucleic acid and protein coat. Thus, all vital activities must depend on the host cell, i.e. the expansion of the virus must depend on the host cell.
The canine kidney (Madin-Darby canine kidney cells, MDCK) cells are established by separating and culturing Madin and Darby, and have the advantages of easy culture, fast proliferation, high infection efficiency of influenza virus and the like, and can be used for amplifying the virus.
However, media and methods for culturing viruses using MDCK cells are currently under investigation.
Disclosure of Invention
The present invention aims to solve, at least to some extent, one of the above technical problems or at least to provide a useful commercial choice. To this end, an object of the present invention is to propose a method capable of efficiently using a low serum MDCK cell culture medium to culture MDCK cells in an adherent manner and combining a virus maintenance solution to amplify viruses.
The present application has been completed based on the following findings by the inventors:
the traditional MDCK cell culture mode is to carry out adherent growth in MEM culture medium containing about 10% of serum, and the serum can provide required hormone, growth factors, transfer proteins and other nutrients for the growth and proliferation of cells. However, serum has complex components and quality differences between different production places and batches, so that the large-scale cell culture process is adversely affected. In addition, in the process of culturing cells using a serum-containing medium to obtain a product, serum becomes a major obstacle for separation and purification, and the entry of residual serum into a human body inevitably causes unnecessary immune reactions.
In view of the above, the inventors have combined the abundant experience accumulated in the cell culture field before and carried out a lot of screening and optimizing work, and unexpectedly obtained an additive added to MEM culture medium on the premise of reducing the serum usage, and the MEM culture medium added with the additive can complete acclimatization adaptation to MDCK cells more quickly under the condition of low serum, support high-density adherent culture of MDCK cells in a fixed bed bioreactor, have high cell activity rate and low agglomeration rate, can obtain excellent performance on multiple indexes, and simultaneously improve the virus infection capability, and is beneficial to virus infection. Furthermore, in order to enable the virus to amplify better after infecting cells, the inventor obtains a virus maintenance solution through a large number of experimental screening and optimization, and the virus after infecting cells can be effectively amplified in the virus maintenance solution, so that the virus titer is improved, and the method is better applied to the amplification production of vaccine viruses, and the production efficiency is improved.
To this end, in one aspect of the invention, the invention proposes a combinatorial medium for amplifying viruses. According to an embodiment of the present invention, the combined medium for amplifying viruses includes: a culture medium for the adherent culture of MDCK cells; a virus maintenance solution; wherein, the culture medium for the adherent culture of MDCK cells comprises: MEM medium; 3-5% by volume of serum; an additive, the additive comprising: 65.2 parts by weight of L-alanine; 55.384 parts by weight of L-arginine hydrochloride; 11.08 parts by weight of L-aspartic acid; 27.04 parts by weight of L-asparagine; 13 parts by weight of L-cysteine hydrochloride monohydrate; 20 parts by weight of L-cystine dihydrochloride; 75.944 parts by weight of L-glutamic acid; 105.784 parts by weight of L-glutamine; 36.928 parts by weight of glycine; 5.704 parts by weight of L-histidine hydrochloride monohydrate; 22.4 parts by weight of L-hydroxyproline; 15.92 parts by weight of L-isoleucine; 20.4 parts by weight of L-leucine; 21.472 parts by weight of L-lysine hydrochloride; 12.944 parts by weight of L-methionine; 7.872 parts by weight of L-phenylalanine; 40.424 parts by weight of L-proline; 39.184 parts by weight of L-serine; 6.224 parts by weight of L-threonine; 4.144 parts by weight of L-tryptophan; 14.44 parts by weight of L-valine; 695.2 parts by weight of alanylglutamine; 83 parts by weight of sodium pyruvate; 960 parts by weight of HEPES;640 parts by weight of soybean hydrolysate; 640 parts by weight of yeast hydrolysate; 196.4 parts by weight of anhydrous disodium hydrogen phosphate; 1590.384 parts by weight of D-glucose anhydrous; 0.208 parts by weight of reduced glutathione; 0.248 parts by weight of ferrous sulfate heptahydrate; 0.272 parts by weight of zinc sulfate heptahydrate; 1.2 parts by weight of vitamin C;13.32 parts by weight of choline chloride; 0.504 parts by weight of calcium D-pantothenate; 0.46 parts by weight of folic acid; 13.4424 parts by weight of inositol; 0.02 parts by weight of ferric nitrate nonahydrate; 0.5 parts by weight of lithium chloride; 2.2 parts by weight of citric acid; 4 parts by weight of taurine; 0.8 parts by weight of recombinant human insulin; 4 parts by weight of ferric citrate; 0.96 parts by weight of ethanolamine; 0.84 parts by weight of EDTA;1.112 parts by weight of nicotinamide; 1.0344 parts by weight of vitamin B6;0.328 parts by weight of vitamin B1;2.552 parts by weight of vitamin B12;6.4 parts by weight of anhydrous L-tyrosine disodium salt; 0.00104 parts by weight of copper sulfate pentahydrate; 33.52 parts by weight of magnesium chloride hexahydrate; 0.072 parts by weight of D-biotin; 1.432 parts by weight of hypoxanthine; 0.0496 parts by weight of linoleic acid; 0.168 parts by weight of lipoic acid; 0.048 parts by weight of 1, 4-butanediamine dihydrochloride; 0.104 parts by weight of vitamin B2;0.312 parts by weight of thymidine; 0.208 parts by weight of p-aminobenzoic acid; 0.00016006 parts by weight of aluminum chloride; 0.000460172 parts by weight of barium acetate; 0.000200075 parts by weight of cobalt chloride hexahydrate; 1.6006E-05 parts by weight of manganese chloride tetrahydrate; 0.000600224 parts by weight of sodium fluoride; 0.000140052 parts by weight of stannous chloride; 0.000140052 parts by weight of cadmium chloride; 6.00224E-05 parts by weight of germanium dioxide; 0.004 parts by weight of sodium selenite; 0.000800299 parts by weight of sodium metasilicate nonahydrate; 2.00075E-05 parts by weight of nickel chloride; 4.00149E-05 parts by weight of ammonium metavanadate; 5.00E-06 parts by weight of potassium bromide; 1.6006E-05 parts by weight of potassium iodide; 8.00E-07 parts by weight of rubidium chloride; 1.00037E-05 parts by weight of ammonium molybdate; 6.00E-07 parts by weight of silver nitrate; 0.06 parts by weight of adenine; 0.02 parts by weight of guanine hydrochloride; 0.01 parts by weight of vitamin D2;0.1328 parts by weight of vitamin E;0.016 parts by weight of vitamin A acetate; 0.0024 parts by weight of 5-amino-4-carboxamide imidazole; 0.004 parts by weight of nicotinic acid; 0.06 parts by weight of 2D-deoxyribose; 0.0008 parts by weight of hydrocortisone; 0.016184561 parts by weight of myristic acid; 0.016184561 parts by weight of oleic acid; 0.016184561 parts by weight of palmitic acid; 0.016184561 parts by weight of stearic acid; 0.0048 parts by weight of EGF;0.0024 parts by weight of IGF; the virus maintenance solution comprises: the culture medium for the adherent culture of MDCK cells does not contain soy hydrolysate, yeast hydrolysate, EGF and IGF.
According to the examples of the present application, the inventors of the present application found that the versatility of the culture conditions applied to different cells is poor, and that different culture methods need to be developed based on the growth characteristics of the cells themselves in order to achieve efficient cell expansion or culture. Furthermore, the inventor performs a great deal of optimization and screening experiments aiming at the characteristics of MDCK cells, obtains the additive, can realize the domestication adaptation of MDCK cells by culturing the MDCK cells under the condition of low serum by adding the additive into a MEM culture medium, supports the high-density adherent culture of the MDCK cells in a fixed bed bioreactor, has high cell activity rate and low agglomeration rate, can obtain excellent performance on a plurality of indexes, improves the virus infection capability, and is beneficial to virus infection. Furthermore, in order to enable better amplification after virus infection of cells, the inventor obtains the virus maintenance solution through a large number of experimental screening and optimization, and soybean hydrolysate, yeast hydrolysate, EGF and IGF are reduced in a culture medium for wall-attached culture of MDCK cells, so that the MDCK cells can grow, the virus amplification is facilitated, the virus titer is improved, and the virus maintenance solution is better applied to vaccine virus amplification production, and the production efficiency is improved.
In another aspect of the invention, the invention provides a method of amplifying a virus, the method comprising, according to an embodiment of the invention: performing adherence culture on MDCK cells by using a culture medium for adherence culture on MDCK cells in the combined culture medium for amplifying viruses; collecting the cells obtained by the culture and adding the cells into the virus maintenance solution in the combined culture medium for amplifying viruses to obtain cell solution; the virus is inoculated into the cell fluid for culture so as to amplify the virus. Therefore, the MDCK cell culture solution obtained by culturing by adopting the method disclosed by the embodiment of the invention is beneficial to realizing virus proliferation and improving the virus titer, so that the method is better applied to the amplification production of vaccine viruses and improves the production efficiency.
According to an embodiment of the invention, the virus is selected from seasonal influenza viruses such as influenza a virus H1N1, H3N2, influenza b virus BY, BV, etc.
In yet another aspect of the invention, the invention provides a method of preparing a vaccine. According to an embodiment of the invention, the method comprises: viruses were amplified according to the methods for amplifying viruses described above. Therefore, the vaccine with high virus titer and high yield can be obtained by using the method provided by the embodiment of the invention, and the preparation method is simple and convenient to operate, high in productivity and suitable for large-scale production and application.
According to an embodiment of the invention, the method further comprises: subjecting the virus solution obtained by amplifying the virus to an attenuation or inactivation treatment so as to obtain the vaccine. Thus, adverse reactions caused by viruses entering the body can be avoided.
According to an embodiment of the invention, the vaccine is selected from the group consisting of a human seasonal influenza vaccine, an avian influenza vaccine, a swine influenza vaccine.
The term "vaccine" refers to a composition containing an active ingredient effective to induce a degree of immunity against a pathogen or disease in a subject that will cause at least a reduction (up to complete lack) in the severity, duration or other manifestation of symptoms associated with infection by the pathogen or disease. The subject is preferably a mammal, such as a pig, cow, sheep, bird, mouse or human.
In yet another aspect of the invention, the invention provides a vaccine. According to an embodiment of the invention, the vaccine is obtained by the method of preparing a vaccine as described previously. Therefore, the vaccine provided by the embodiment of the invention is safe and effective, has small adverse reaction after inoculation, and is suitable for popularization and application.
In a further aspect of the invention, the invention provides the use of a vaccine as described hereinbefore in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment or prevention of a virus-related disease.
According to an embodiment of the invention, the virus-related disease is selected from the group consisting of human seasonal influenza, avian influenza or swine influenza vaccines.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a graph of a total cell count analysis of MDCK cells serially passaged in different media in accordance with one embodiment of the present invention;
FIG. 2 shows a graph of analysis of the serial multiplication time of MDCK cells in different media, according to one embodiment of the invention;
FIG. 3 shows graphs of toxicity titers analysis using different maintenance fluids after MDCK inoculation with H1N1 virus according to one embodiment of the present invention.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were not specific to the manufacturer and were conventional products commercially available, wherein pea hydrolysate (schkai organism, cat No. a 2501), soybean hydrolysate (schkai organism, a 1603), yeast hydrolysate (schkai organism, a 1202), recombinant human insulin (Tongdongbao).
Example 1
1. Preparing culture medium and virus maintenance liquid
1.1 MEM medium
Figure 55297DEST_PATH_IMAGE001
1.2 SLM1 medium and SLM2 medium
3% NBS was added to MEM medium and the following ingredients were added separately:
Figure 965484DEST_PATH_IMAGE002
Figure 421873DEST_PATH_IMAGE003
Figure 177340DEST_PATH_IMAGE005
1.3 Commercial culture medium
Gibco (cat. No. 12559027) was formulated according to instructions.
1.4 Virus maintenance liquids A1 and A2
Virus maintenance liquids A1 and A2 corresponding to the SLM1 medium and the SLM2 medium were respectively prepared, except that soybean hydrolysate, yeast hydrolysate, recombinant human insulin, EGF and IGF were not contained.
1.5 Virus maintenance solution A3
The composition was the same as that of SLM1 culture medium.
2. Cell culture and detection
MDCK cells were subjected to adherent culture with SLM1, SLM2, commercial medium, MEM+10% NBS prepared as described above in the following manner: MDCK cells were cultured in 6X 10 cells 6 Inoculating the cell amount into a T175 square bottle, placing the T175 square bottle in a 5% carbon dioxide incubator at 37 ℃ for static culture, inoculating the T175 square bottle into a new culture bottle according to the cell amount for continuous passage after 48 hours, recording the total cell amount of the culture for 48 hours, and calculating the multiplication time of MDCK cells cultured in different culture media.
The total cell amount and the cell multiplication time in the passage process of different culture mediums are shown in figures 1 and 2, and in the continuous passage process, the total cell amount SLM1 of 48 hours is more than commodity culture medium is more than SLM2 is more than MEM+10% NBS; cell doubling time SLM1 < commercial medium < SLM2 < MEM+10% NBS. The low serum culture medium SLM1 has a culture effect obviously superior to that of a serum culture medium, and has a growth speed faster than that of a commercial culture medium, so that the high-density growth of MDCK is satisfied.
3. Virus inoculation and virus titer detection
Square bottles cultured by using culture mediums of SLM1, SLM1 and SLM2 are respectively washed twice by using culture solution without serum after cells are full of a single layer, A1, A3 and A2 virus maintenance solutions are respectively replaced, influenza virus (H1N 1) is inoculated according to MOI of 0.001, and sampling and detecting hemagglutination titer HA are carried out 24H and 48H after virus inoculation. As shown in FIG. 3, the hemagglutination titers of the SLM1 and the A1 are superior to those of other combinations, and after MDCK cells are infected with viruses, the MDCK cells can be effectively amplified in the virus maintenance solution, so that the virus titers are improved.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (7)

1. A combination medium for amplifying a virus, comprising:
a culture medium for the adherent culture of MDCK cells; and
a virus maintenance solution;
wherein, the composition of the culture medium for the adherent culture of MDCK cells is as follows:
MEM medium;
3-5% by volume of serum; and
the composition of the additive is as follows:
l-alanine at a final concentration of 65.2 mg/L; l-arginine hydrochloride at a final concentration of 55.384 mg/L; l-aspartic acid with the final concentration of 11.08 mg/L; l-asparagine at a final concentration of 27.04 mg/L; l-cysteine hydrochloride monohydrate at a final concentration of 13 mg/L; l-cystine dihydrochloride at a final concentration of 20 mg/L; l-shaped material with final concentration of 75.944mg/LGlutamic acid; l-glutamine at a final concentration of 105.784 mg/L; glycine with final concentration of 36.928 mg/L; l-histidine hydrochloride monohydrate at a final concentration of 5.704 mg/L; l-hydroxyproline at a final concentration of 22.4 mg/L; l-isoleucine at a final concentration of 15.92 mg/L; l-leucine at a final concentration of 20.4 mg/L; l-lysine hydrochloride with a final concentration of 21.472 mg/L; l-methionine in a final concentration of 12.944 mg/L; l-phenylalanine at a final concentration of 7.872 mg/L; l-proline at a final concentration of 40.424 mg/L; l-serine at a final concentration of 39.184 mg/L; l-threonine with a final concentration of 6.224 mg/L; l-tryptophan with a final concentration of 4.144 mg/L; l-valine at a final concentration of 14.44 mg/L; alanylglutamine with a final concentration of 695.2 mg/L; sodium pyruvate with a final concentration of 83 mg/L; HEPES at a final concentration of 960 mg/L; soybean hydrolysate with a final concentration of 640 mg/L; a final concentration of 640mg/L yeast hydrolysate; anhydrous disodium hydrogen phosphate with a final concentration of 196.4 mg/L; d-glucose anhydrous with a final concentration of 1590.384 mg/L; reduced glutathione at a final concentration of 0.208 mg/L; ferrous sulfate heptahydrate with a final concentration of 0.248 mg/L; zinc sulfate heptahydrate with a final concentration of 0.272 mg/L; vitamin C with a final concentration of 1.2 mg/L; choline chloride with a final concentration of 13.32 mg/L; calcium D-pantothenate at a final concentration of 0.504 mg/L; folic acid with the final concentration of 0.46 mg/L; myo-inositol at a final concentration of 13.4424 mg/L; ferric nitrate nonahydrate with a final concentration of 0.02 mg/L; lithium chloride with a final concentration of 0.5 mg/L; citric acid with a final concentration of 2.2 mg/L; taurine with a final concentration of 4 mg/L; recombinant human insulin at a final concentration of 0.8 mg/L; ferric citrate with a final concentration of 4 mg/L; ethanolamine with the final concentration of 0.96 mg/L; EDTA at a final concentration of 0.84 mg/L; nicotinamide at a final concentration of 1.112 mg/L; vitamin B6 with a final concentration of 1.0344 mg/L; vitamin B1 at a final concentration of 0.328 mg/L; vitamin B12 with a final concentration of 2.552 mg/L; anhydrous L-tyrosine disodium salt with a final concentration of 6.4 mg/L; copper sulfate pentahydrate with a final concentration of 0.00104 mg/L; magnesium chloride hexahydrate with a final concentration of 33.52 mg/L; d-biotin at a final concentration of 0.072 mg/L; hypoxanthine with a final concentration of 1.432 mg/L; linoleic acid with final concentration of 0.0496 mg/L; lipoic acid with a final concentration of 0.168 mg/L; 1, 4-butanediamine dihydrochloride at a final concentration of 0.048 mg/L; vitamin B2 at a final concentration of 0.104 mg/L; thymidine with a final concentration of 0.312 mg/L; final concentration of0.208mg/L of p-aminobenzoic acid; aluminum chloride with a final concentration of 0.00016006 mg/L; barium acetate with a final concentration of 0.000460172 mg/L; cobalt chloride hexahydrate with a final concentration of 0.000200075 mg/L; final concentration of 1.6006X 10 -5 mg/L manganese chloride tetrahydrate; sodium fluoride with a final concentration of 0.000600224 mg/L; stannous chloride at a final concentration of 0.000140052 mg/L; cadmium chloride with a final concentration of 0.000140052 mg/L; final concentration of 6.00224X 10 -5 mg/L germanium dioxide; sodium selenite with final concentration of 0.004 mg/L; sodium metasilicate nonahydrate with a final concentration of 0.000800299 mg/L; final concentration of 2.00075X 10 -5 mg/L nickel chloride; final concentration of 4.00149X 10 -5 mg/L ammonium metavanadate; final concentration of 5.00X 10 -6 mg/L potassium bromide; final concentration of 1.6006X 10 -5 mg/L potassium iodide; final concentration of 8.00X 10 -7 mg/L rubidium chloride; final concentration of 1.00037X 10 -5 mg/L ammonium molybdate; final concentration of 6.00X 10 - 7 mg/L silver nitrate; adenine in a final concentration of 0.06 mg/L; guanine hydrochloride with a final concentration of 0.02 mg/L; vitamin D2 at a final concentration of 0.01 mg/L; vitamin E with a final concentration of 0.1328 mg/L; vitamin A acetate with final concentration of 0.016 mg/L; 5-amino-4-carboxamide imidazole having a final concentration of 0.0024 mg/L; nicotinic acid with the final concentration of 0.004 mg/L; 2D-deoxyribose with a final concentration of 0.06 mg/L; hydrocortisone at a final concentration of 0.0008 mg/L; myristic acid at a final concentration of 0.016184561 mg/L; oleic acid at a final concentration of 0.016184561 mg/L; palmitic acid with a final concentration of 0.016184561 mg/L; stearic acid at a final concentration of 0.016184561 mg/L; EGF at a final concentration of 0.0048 mg/L; IGF with a final concentration of 0.0024 mg/L;
the virus maintenance solution comprises: the culture medium for the adherent culture of MDCK cells does not contain soy hydrolysate, yeast hydrolysate, EGF and IGF.
2. A method of amplifying a virus, comprising:
performing adherent culture on MDCK cells using the medium for adherent culture of MDCK cells in the combined medium for amplifying viruses according to claim 1;
collecting the cells obtained by the culture and adding the cells to the virus maintenance solution in the combined culture medium for amplifying viruses according to claim 1 to obtain a cell solution;
the virus is inoculated into the cell fluid for culture so as to amplify the virus.
3. The method of claim 2, wherein the virus is selected from seasonal influenza viruses.
4. A method according to claim 3, wherein the seasonal influenza virus comprises influenza a virus H1N1, H3N2, influenza b virus BY, BV.
5. A method of preparing a vaccine comprising: the method of claim 2 or 3, wherein the virus is amplified.
6. The method as recited in claim 5, further comprising:
subjecting the virus solution obtained by amplifying the virus to an attenuation or inactivation treatment so as to obtain the vaccine.
7. The method of claim 5, wherein the vaccine is selected from the group consisting of a human seasonal influenza vaccine, an avian influenza vaccine, and a swine influenza vaccine.
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